This study was conducted to determine how 3D cultured trophoblasts' secreted factors impact NK-92 activation and cytotoxicity during early pregnancy. In this study, 6 week gestational age human cytotrophoblast stem cells (iCTB) were cultured in 2D, 3D matrigel, and 3D synthetic hydrogels composed of 20 kDa 4-arm poly(ethylene glycol)-maleimide (PEG-Mal) modified with a GFOGR adhesive ligand (1 mM) and crosslinked with dithiothreitol (DTT), a non-degradable crosslinker. On day six, trophoblast supernatants were collected to investigate the influence of trophoblast organoid secreted factors on activated NK cell phenotype, measured by CD107a expression and levels of IFNγ secretion. Here we demonstrate that NK-92 cells possess a dNK2-like phenotype, and that supernatants of cytotrophoblasts cultured in 2D and synthetic hydrogels, but not matrigel, reduce activated NK-92 cytokine secretion.

The current clinical gold standards for tissue sealing include sutures, staples, and glues, however several adverse effects limit their use. Sutures and staples inherently cause additional trauma to tissue surrounding the wound, and glues can be lacking in adhesion and are potentially inflammatory. All three also introduce risk of infection. Light-activated tissue sealing, particularly the use of near-infrared light, is an attractive alternative, as it localizes heat, thereby preventing thermal damage to the surrounding healthy tissue. Previous work identified a glutaraldehyde-crosslinked chitosan film as a lead sealant for gastrointestinal incision sealing, but in vivo testing resulted in tissue degradation in and around the wound. The suggested causes for this degradation were excess acetic acid, endotoxins in the chitosan, and thermal damage. A basic buffer wash protocol was developed to remove excess acid from the films following fabrication. UV-Vis spectroscopy demonstrated that following the wash, films had the same concentration of Indocyanine green as unwashed films, allowing them to absorb light at the same wavelength, therefore showing the wash did not affect the film’s function. However subsequent washes led to degradation of film mass of nearly 20%. Standard chitosan films had significantly greater mass gain (p = 0.028) and significantly less subsequent loss (p= 0.012) than endotoxin free chitosan-films after soaking in phosphate buffered saline for varying durations , while soaking duration had no effect (p = 0.332). Leak pressure testing of films prepared with varying numbers of buffer washes, laser temperature, and lasering time revealed no significant interaction between any of the 3 variables. As such, it was confirmed that proceeding with in vivo testing with the buffer wash, various lasering temperatures, and laser times would not affect the sealing performance of the films. Future investigation will involve characterization of additional materials that may be effective for sealing of internal wounds, as well as drug loading of agents that may hasten the healing process.